All publications, in particular US patents and patent applications, cited in this specification are incorporated by reference in their entirety.
Biodegradable materials which are useful as temporary artificial ECMs have been developed over the past decades (Yang et al Tissue Engineering 2001 7(6) 679-689). Their usefulness rely among others on their biocompatability and resemblance to natural ECMs (Hoffmann, Advanced Drug Delivery Reviews 2002 54(1) 3-12).
Prestwich et al. pioneered modification of HA by thiolation (Shu et al Biomacromolecules 2002 3 1304-1311) making it capable to cross-link with PEG diacrylate (Shu et al Biomaterials 2004 25 1339-1348) or PEG bis-maleimide (Vanderhooft et al Biomacromolecules 2007 8 2883-2889) for in situ production of hydrogel scaffolds that are degraded by hyaluronidase. Gelation of thiolated HA with RGD-peptide containing tri-block acrylate-PEG-CC(PEG-acrylate) RGDS cross-linker (Shu. et al Biomed Mater. Res. 2004, 68(A) 365-375) or combination of thiol-modified HA with thiol-modified gelatin (Shu et al J. Biomed. Mater. Res. 2006 79(A) 902-912; and Prestwich et al Advan. Enzyme Regul. 2007 47 196-207) allowed to construct cytoadherent synthetic ECM mimics for tissue engineering. An alternative functionalization, when electrophilic acrylate groups are localized on HA, whereas the sulfhydryl groups on PEG (Kim et al Current Applied Physics 2007, 7(S1) e28-e32; and Kim et al Biomaterials 2007 28 1830-1837) or bis-cysteine peptide cross-linker (Hahn et al Int. J. Pharm. 2006 322 44-51) was also examined for hydrogel preparation by Michael addition. Formation of reversible thiazolidine cross-linkages between cysteine 1,2-aminothiol terminated dendron and PEG dialdehyde was used to render a hydrogel that stays intact for approximately one week after preparation (Wathier et al J. Am. Chem. Soc. 2004 126 12744-12745). Finally hydrazone formation is one of a few other examples of cross-linking chemistries that were suggested for in vivo production of hydrogel materials (Luo et al J. Control. Release 2000 69 169-184; Kirker et al J. Polym. Sci. Part B 2004 42 4344-4356; and Kirker et al Biomaterials 2002 23 3661-3671). Thus, both hydrolytically (due to hydrolysis of hydrazone linkages) and enzymatically degradable hydrogels were prepared from hydrazide-modified HA and PEG dialdehyde (Luo et al J. Control. Release 2000, 69, 169-184; and Kirker et al J. Polym. Sci. Part B 2004, 42, 4344-4356) and used for wound healing (Kirker et al Biomaterials 2002 23 3661-3671).
A limited number of cross-linkers with suitable reaction kinetics are available for preparation of hydrogels, e.g. by changing the reactivity of thiophilic bis-functional cross-linkers (Vanderhooft et al Biomacromolecules 2007, 8, 2883-2889). Another approach is the use of multifunctional cross-linkers which was recently realized for HA by its modification into two HA derivatives comprising side chains terminated with electrophilic aldehyde and nucleophilic hydrazide chemoselective functionalities respectively (Bulpitt et al Aeschlimann, D. J. Biomed. Mater. Res. 1999 47 152-169; Jia et al Biomaterials 2004, 25, 4797-4804; and Jia et al Biomacromolecules 2006 7 3336-3344. This topic has also been investigated by us (Ossipov et al J. Appl. Polymer Sci. 2007 106 60-70).
Other examples of publications dealing with the field of the invention are Aeschlimann et al. (U.S. Pat. No. 7,196,180; U.S. Pat. No. 6,630,457 and US 20070149441; Bulpitt et al. U.S. Pat. No. 6,620,927 and U.S. Pat. No. 6,884,788; Park et al Biomaterials 24 (2003) 893-900; Spiro et al U.S. Pat. No. 6,303,585; and Crescenzi et al Biomacromolecules (2007 No 8 1844-1858).
Formation of hydrogels in situ by cross-linking synthetic polyhydroxypolymers by the use of homo-multifunctional cross-linking reagents that comprises non-biopolymer structure has also been reported (Ossipov et al J. Appl. Polymer Sci. 2007 106 60-70; Schmedlen et al Biomaterials 2002 23 4325-4332; and Reyes et al Ophthalmology & Visual Sci. 2005 46(4) 1247-1250.
A search performed by the Swedish Patent Office with respect to the SE priority application has cited Prestwich et al (US 20080032920) which deals with hydrogel formation by reacting a thiolated macromolecule with an α-halo carbonyl modified macromolecule.
Unfavourable results with respect to biocompatability have been reported for amino-derivatized hyaluronic acid cross-linked with an uncharacterized aldehyde-modified dextran (Bulpitt et al J. Biomed. Mat. Res. 47(2) (1999) 152-169).
Bergman et al (Biomacromolecules 8(7) (2007) 2190-2195) have reported about the versatility of triazine activation of the carboxy group of hyaluronic acid.
Objectives
Hydrogels that are formed in situ from polymeric networks based on cross-linking of multifunctional polymers should be implantable by minimally invasive surgery. Such polymers including precursors of the network should thus be possible to administer through syringes, e.g. by injection. This mode of administration typically requires that the precursor reagents during the administration are present as a mixture in a liquid media which should have viscosity that is low enough to permit easy administration through a syringe used and enabling the liquid to easily fill complex shaped areas with good contact for the formed hydrogel to the native tissue. Cells and/or bioactive substances, such as therapeutic agents, should easily be able to penetrate or be encapsulated in the formed gel See for instance Gutowska et al Anatomical Record 2001 263(4) 342-349; Vargese et al Adv. Polym. Sci. 2006 203 (Polymers for regenerative medicine) 95-144; Hubbell et al Curr. Opin. Solid State and Mat. Sci. 1998 3 246-251; and Lee et al Chem. Rev. 2001 101(7) 169-1879. Similar viscosity criteria apply to the handling of individual formulations of precursor reagents (HA and CLR). In general the viscosity should be low enough to permit quick mixing of the components/reagents in order to achieve rapid cross-linking and hydrogel formation.
Several requirements must be fulfilled for multifunctional polymer systems to be used for the in situ formation of hydrogels by cross-linking in vivo. The systems have to be biocompatible after implantation of the hydrogel, which in addition to biocompatibility of the hydrogel as such means that a) precursor compounds should have the ability to crosslink in vivo without forming toxic by-products, and b) degradation products from the polymeric network of the hydrogel have to be harmless. Cross-linking should take place by utilizing addition reactions and/or substitution reactions and/or elimination reactions each of which should release no or only harmless molecules (e.g. H2O and molecules which as such may be harmful but whose negative effects easily and quickly can be neutralized in vivo). The use of reactants other than the polymer to be cross-linked and the cross-linking reagent should be avoided both from a practical point of view and the fact that such agents also would imply biocompatability requirements similar to those applicable to the starting basic components, i.e. in our case the hyaluronan and the cross-linking reagent. The cross-linking reactions should be selective and appear at a high rate leading to gel formation within less than some minutes (typically less than a minute or two) and yet allow for sufficient mixing of the components (Ossipov et al J. Appl. Polymer Sci. 2007 106 60-70). The gels should be degradable in vivo at a rate that often varies for different applications and effects to be achieved, for instance for a) different tissue regenerations and encapsulated grow factors, b) different immunogens and immunization protocols, c) different tissues to be glued and gluing protocols, d) different bioactive substances, such as drugs or other therapeutic agents, e) to have local and/or systemic effects etc. Thus it is desirable with polymeric systems for which it is possible to control the cross-linking degree as well as the rate of degradation in vivo of the hydrogels, for instance by varying kind, molecular weight and concentration of starting components, kind of reactive functional groups and their concentration in the mixture to be administered, relative amount of reactive counterparts, i.e. (total amount of reactive HA-groups)/(total amount of reactive CLR-groups), degree of substitutions with respect to (content of) reactive HA-group and reactive CLR group in the hyaluronan and the cross-linking reagent, respectively, etc.
The use in vivo of gels based on synthetic polymers and other non-endogenous polymeric materials is restricted by the risk of bio-incompatability, i.e. the risk of eliciting a negative body response, for instance inflammatory and anaphylactic responses of either immunological or non-immunological origin. The level of such responses for the gels as such and precursor reagents should be kept at an acceptable level or at a level not observable in test models and systems as is well-known in the literature. Thus infiltration of inflammatory cells, including giant cells and lymphocytes, should be insignificant, i.e. be void or non-indicative of chronic inflammation. In vitro cytotoxicity tests should show insignificant cytotoxicity.
A general objective and challenge thus is to provide polymer based systems for in situ formation of hydrogels meeting one or more of the above-mentioned requirements.
The Invention
The present inventors have realized that the above-mentioned challenge at least partly can be coped with by using a cross-linking reagent which provides a cross-linking structure which exhibits a plurality of hydroxyl groups and is devoid of hyaluronan structure.
In one of its main aspects (first aspect) the present invention thus is a composition containing a hyaluronan-formulation (HA-formulation) and a formulation of a homo-multifunctional cross-linking reagent (CLR-formulation) which is devoid of hyaluronan structure and provides a plurality of hydroxyl groups in the cross-linking structure introduced by the reagent. The hyaluronan of the HA-formulation is cross-linkable by the use of the homo-multifunctional cross-linking reagent. Plurality for number of hydroxy groups in the cross-linking reagent and/or cross-linking structure preferably mean≧5 or ≧10. The composition is activatable in the sense that the cross-linking reagent and the hyaluronan are initially unreactive towards each other but upon activation of the composition cross-linking spontaneously starts in an aqueous liquid with formation in situ of the hydrogel. The aqueous liquid contains at the start of the cross-linking (=end of activation) active forms of both the hyaluronan and the cross-linking reagent. The cross-linking reaction is a selective reaction between a reactive functional group of the cross-linking reagent and a reactive functional group of the hyaluronan. The two reactive groups are counterparts to each other as defined above (reactive CLR-group and reactive HA-group, respectively).
A characterizing feature of preferred variants of the composition is that it comprises:    A) a HA-formulation in which the hyaluronan exhibits the carbohydrate backbone of native hyaluronic acid (HA-structure/HA-backbone) and a plurality of a reactive substituent (reactive HA-substituent) which            i) is directly attached to the HA-backbone,        ii) exhibits a reactive HA-group as defined above, and            B) a CLR-formulation containing a multi-homofunctional polyhydroxy-containing cross-linking reagent, which comprises a plurality of a reactive CLR-group as defined above.
The reactive HA-group is absent in native hyaluronic acid. The cross-linking reagent is devoid of hyaluronan structure, i.e. has a backbone structure that is different from the backbone structure of native hyaluronic acid.
The cross-linking reagent is in preferred variants a polyhydroxy polymer (PHP) which comprises a backbone structure (PHP-backbone) and a plurality of the reactive CLR-substituent which is directly attached to the PHP-backbone and exhibits the reactive CLR-group. The abbreviation PHP will be used instead of CLR for variants in which the cross-linking reagent imperatively is a polyhydroxypolymer.
The hyaluronan and the cross-linking reagent are preferably water-soluble, e.g. at physiologically acceptable pH conditions as defined elsewhere in this specification and at a temperature within the interval of 15-45° C., possibly together with a buffer. This in particular applies to forms of the reagents in which the reactive counterparts are in active form, e.g. lacking protecting groups.
With respect to biocompatibility the composition is characterized in that the hyaluronan and the cross-linking reagent have been selected so that the hydrogel formed as a consequence of activation and cross-linking is biocompatible by complying with the general rules outlined under the heading “Objectives”. Thus a gel produced according to the invention that is devoid of a bioactive stubstance of the types described herein as additive should be characterized in giving an acceptable response with respect to foreign body giant cell reaction indicative of chronic inflammation in the in vivo experiments used by Bulpitt et al. (J. Biomed. Mat. Res. 47(2) (1999) 152-169) and/or by us in the experimental part of this specification. Thus, infiltration of inflammatory cells, including giant cells and/or lymphocytes, should preferably be unobservable. Cytotoxicity, for instance measured in vitro as done in the experimental part, should be insignificant, e.g. within ±75%, such as ±50%, of the value obtained for the controls (that should be of the same kind as in our experimental part).
Biocompatability of the kinds discussed in the preceding paragraph will depend on factors controlling penetratability of the formed gel by inflammatory cells with a high penetratability permitting more infiltration. The cross-linking degree and groups/structures of the final gel are for instance of importance. Thus by proper selection of kind and amount of cross-linking reagent and its reactive CLR-groups in relation to the hylauronan and its reactive HA-groups the above-mentioned biocompatibility can be accomplished. Testing/screening for appropriate combinations of reagents is done in the model systems given.
An alternative or a complement to the above-mentioned kind of biocompatability is an official approval issued by an ethical committee at a university, hospital and the like and/or by a regulatory authority in one, two or more countries selected amongst the US, JP, EPO-countries for using a gel produced according to the invention in testing in animals, such as humans, and/or in therapy. EPO-countries of importance are Great Britain, the Netherlands, Belgium, France, Spain, Portugal, Italy, Switzerland, Austria, Luxembourg, Germany, Sweden, Denmark, Finland etc.
The HA-formulation and the CLR-formulation of the inventive composition shall support suitable conditions for quick cross-linking and in situ hydrogel formation within given time frames in the aqueous solution resulting from the activation of the composition. Suitable time frames as measured in vitro according to the method given in the experimental part are ≦15 minutes, such as ≦10 minutes with preference for ≦5 or ≦4≦3 minutes and with the highest preference for ≦2 or ≦1 minute. The lower end of these intervals is typically 5 seconds or 10 seconds or 15 seconds.
Useful desired elastic modulus values (shear storage modulus, G′) of suitable hydrogels will depend on the intended application and can therefore be found in a broad interval, such as 10 Pa to 1 MPa, most likely with preference for 100 Pa to 10 kPa. Measurement is according to the method given in the experimental part.
Values for the swelling ability of suitable cross-linked hyaluronans hydrogels will depend on the intended application and can therefore be found in a broad interval, such as ≧0%, e.g. ≧10%, and/or ≦10000%, e.g. ≦1000%. Measurement is according to the method given in the experimental part.
Testing/screening for suitable cross-linking conditions that will give hydrogels having gelation times, elastic modulus, and swelling ability that fit predetermined values within the intervals given can be carried out in vitro by methods given in the experimental part. In this kind of screening, parameters that are tested can be selected amongst pH, cross-linking reagents (kind and type), pairs of reactive counterparts including degrees of substitution, total and/or relative concentrations, size and form (e.g. Mw) of hyaluronan and/or cross-linking reagent etc. Screening/testing means that two or more experiments that differ with respect to at least one parameter are carried out and the results compared in order to determine favourable conditions.
Suitable rates of disappearance (resorbability, degradation) of the gel in vivo will depend highly on the particular application and will thus be found within a wide interval, for instance between 1-2 days up to a year for the gel to fully disappear. Thus in the case of cosmetic surgery it may be beneficial for the individual undergoing the treatment if the gel disappears/is resorbed within 2-24 months or a longer period of time with preferred values being less than 12 months. For medical treatments the gel should remain until the effect desired for the dose given has been accomplished. For tissue generations this means as long as it takes for the desired generation to occur and/or if a bioactive substance is incorporated in the gel as long as there is active substance in the gel and/or the desired effect for the dose given has been accomplished. As a general guideline suitable gels can be found amongst those gels which when prepared in vivo as a consequence of subcutaneous or intramuscular injection in rats of an activated composition according to the invention will be retrievable at the location for the administration for a period of time that is within the interval of 1 day to 12 months, such as ≧1 week or ≧1 month or/or ≦8 months or ≦6 months with measurements taking place in the animal model as given in the experimental part including doses given.
Screening/testing for gels giving appropriate disappearance/resorption rates for a desired application is carried by use of the animal model used in the experimental part with screening parameters being in principle the same as for the above-mentioned in vitro screening.
Activation of the Composition, Cross-Linking and Hydrogel Formation
A reactive CLR-group and/or a reactive HA-group may be present in the composition in protected (=activatable/inactive) form or in active form, where protected form means that the group can be transformed (=activated) to the active form which reacts with its counterpart (if in active form) to form the above-mentioned linkage structure. The term “protected” is used in a broad sense and encompasses a) physical separation of the hyaluronan and the cross-linking reagent from each other, and also b) chemical protection, i.e. the reactive group comprises one or more chemical groups that hinder the cross-linking reaction (=protecting groups). Alternative (a) comprises e.g. that the HA- and the CLR-formulations are present in different compartments of the composition or in separate physical states, for instance different powder forms and different other dry or dehydrated forms. In the latter case the two formulations may be present in the same compartment of the composition. Typical chemical protecting groups used in alternative (b) are well-known in synthetic chemistry and are typically removable under mild conditions only affecting desired parts of the reagent carrying the protected group. Typical deprotecting conditions useful for the invention encompass for instance changes in pH, temperature, ionic strength etc and may include an increase in the concentration of a particular anion, such as an anionic nucleophile, or a particular cation, such as a cationic electrophile, and neutral nucleophiles and electrophiles. Proper matching of changes in conditions with the protecting group to be removed is normally required.
The hyaluronan and the cross-linking reagent of the composition are upon activation forming the hydrogel in situ by the cross-linking reaction in the aqueous liquid. In variants utilizing cross-linking reagents that are polyhydroxypolymers the cross-linking structure will exhibit a PHP-backbone.
Activation of the composition comprises transforming reactive HA-groups and reactive CLR-groups that are in protected form in the composition to active forms thereof. Both the hyluronan and the cross-linking reagent will be present in active and dissolved form in a common aqueous solution leading to spontaneous cross-linking of the hyaluronan and formation of the hydrogel in situ via the cross-linking. The transformation typically includes one or more steps:                i) transferring the hyaluronan and the cross-linking reagent, if they are present in separate compartments of the composition, to a common aqueous liquid providing dissolving conditions for both the hyaluronan and the cross-linking reagent, and/or        ii) dissolving any reactant selected amongst the hyaluronan and the cross-linking reagent that is in solid form (e.g. powder form, dehydrated form, dry form etc) in an aqueous liquid providing dissolving conditions for the reactant, and/or        iii) removing a protecting group that possibly is present in a reactive HA-group or in a reactive CLR-group in an aqueous liquid providing conditions for the removal of the protecting group.        
Different combinations of steps (i)-(iii) are possible depending on the particular formulations of the hyaluronan and the cross-linking reagent including presence or absence of protecting groups. In preferred variants, for instance, the hyaluronan and the cross-linking reactant are present in dissolved form in separate aqueous liquid aliquots that in turn are present in different compartments of the composition. The final step in the activation is then provided by mixing the aliquots. If any of the hyaluronan and the cross-linking reactant exhibits a reactive counterpart that comprises a protecting group, the mixed liquid should preferably also provide conditions for the removal of such a protecting group, for instance if the removal is pH-dependent such as for protonated amino-containing groups or COOH/COO−-containing groups.
For compositions adapted for cross-linking and in situ formation of the hydrogel to take place in vivo, the activation should end up in an aqueous liquid containing the two counterpart groups in active forms and providing acceptable conditions for cross-linking as well as for the individual to which the composition is to be administered as discussed elsewhere in this specification.
Reactive Counterpart Groups
The pair of two reactive counterparts and their presence on the hyaluronan and on the cross-linking reagent is selected such that reaction between the two groups during the cross-linking conditions applied will be selective in the sense that linkage structures will preferentially be formed between a reactive HA-group and a reactive CLR-group. The hyaluronan therefore is devoid of counterparts to the reactive HA-groups and the cross-linking reagent is devoid of the counterparts to the reactive CLR-groups. The term “selective” also includes that each of the two counterparts are reacting without significant formation of by-products from competing reactions. In preferred variants there is no need for reagents other than the hyaluronan and the cross-linking reagent, except for buffer systems that may act as deprotection/activation agents by changing the degree of protonation of groups that are to be utilized in the cross-linking.
Reactive HA-groups and reactive CLR-groups should not to any significant degree be counterparts to hydroxy during the cross-linking conditions. This typically means that ≧25%, such as ≧50% or ≧75% or ≧95%, of the reactive HA-groups and/or the reactive CLR-groups are reacting with each other in the intended manner during cross-linking conditions.
The ratio between the molar amount (MHA) of the reactive HA-group in the HA-formulation and the molar amount (MCLR) of the reactive CLR-group in the CLR-formulation, i.e. the ratio MHA/MCLR, is typically within the interval of 0.1-10, such as 0.25-4 or 0.5-2, with preference for essentially in equimolar amounts, i.e. 0.75-1.30 or 0.9-1.1. Ratios deviating from equimolarity may be useful in certain variants of the invention, such as when it is desirable to speed up the consumption of a slow-reacting counterpart or to produce a hydrogel which carries reactive groups (either reactive HA- or reactive CLR-groups). Excessive amounts of a reactive counterpart group may be used for immobilizing a bioactive substance of the kind discussed herein to the hydrogel. Such a bioactive substance shall exhibit a reactive group that is counterpart to the reactive group in excess.
The reaction sequence leading to the above-mentioned linkage structure, which preferably is covalent, preferably comprises a substitution reaction step and/or an addition reaction step, possibly combined with a subsequent elimination reaction step. Ring-forming steps may be included, for instance as a cyclization step subsequent to an elimination step or as an addition step (cycloaddition). Sequences comprising one or more of these steps are preferred since it is often possible to select reactive counterparts that are of sufficient reactivity and selectivity for quick formation of the desired cross-linkages with a minimum formation of harmful by-products as discussed in the objectives above.
In one type of reaction sequence either the reactive HA-group or the reactive CLR-group comprises a nucleophilic group while the remaining one of the two counterparts comprises an electrophilic group.
A nucleophilic counterpart typically comprises a first heteroatom O, N, or S (═X) exhibiting a free electron pair with 0-3 hydrogen attached to the heteroatom and one, two or three organic groups being directly bound to the heteroatom. The organic group typically provides a carbon or a second heteroatom O, N and S directly attached to the first heteroatom X, with the proviso that the second heteroatom should be O, N or S when X═N or S, and N or S when X═O. The preferred nucleophilic counterparts are uncharged during cross-linking. For a nucleophilic counterpart which is a base in an acid-base pair, ≧5%, such as ≧25% or ≧50 or ≧75%, of the total concentration of the acid-base pair should be in base form.
An electrophilic counterpart typically comprises an electron-deficient carbon. In preferred variants the electron-deficient carbon is part of a multiple bond between a) two carbons, or b) between one carbon and one heteroatom selected amongst N, O and S. For addition reactions involving formation of a bond between a nucleophilic counterpart (XH) and an electron-deficient double-bonded carbon, this latter carbon typically carries only hydrogen and/or carbon (i.e. single-bonded to the double-bonded carbon). If the multiple bond contains no heteroatom (i.e. is the double-bonded carbon is part of a carbon-carbon double bond) an electron-withdrawing substituent is typically replacing the single-bonded carbon attached to the electron-deficient carbon (i.e. the electron-withdrawing substituent either comprises the single-bonded carbon or a heteroatom attached directly to the electron-deficient double-bonded carbon). In these addition reactions X of the nucleophilic group (XH) becomes bonded to the electron deficient atom of the multiple bond and hydrogen (H) to the other atom of the multiple bond. For substitution reactions with a nucleophilic counterpart at an electron-deficient double-bonded carbon a suitable leaving group has to be present on the carbon. Preferably the other atom of the double bond is then a double-bonded heteroatom O, S or N. During these substitution reactions the nucleophilic counterpart will displace the leaving group on the electron-deficient double-bonded carbon. Suitable leaving groups are providing a heteratom (O, N or S) directly attached to the electron-deficient double-bonded carbon of the multiple bond.
An addition reaction involving nucleophilic and electrophilic counterparts can be followed by elimination of water (H2O) if the nucleophilic counterpart comprises NH2-bound to carbon or a heteroatom (e.g. O or N) in combination with an electrophilic counterpart comprising double bonded oxygen. The elimination reaction typically results in an enamine or a ketamine structure (>C═N—) which will be particularly favourable when the formed C═N double bond will be part of a conjugated system of multiple bonds and/or links directly to a heteroatom N or S. The corresponding preferences apply for the starting nucleophilic and electrophilic counterparts).
The elimination reaction discussed in the preceding paragraph can be followed by a subsequent cyclization reaction if one of the counterparts comprises an additional nucleophilic group exhibiting a heteroatom X′ (O, N, or S) and a hydrogen (H) (nucleophilic group=—X′H). The nucleophilic group (X′H) then should be placed at a distance of 2-3 atoms from XH or from the carbon-oxygen double bond of the electrophilic group for the reaction to proceed smoothly.
Preferred nucleophilic groups are:                a) capable of giving the linkage structure by addition to a counterpart which comprises a keto or aldehyde group possibly followed by elimination of H2O: hydrazide groups e.g. —CONH2NH2 groups, semicarbazide groups including e.g. semicarbazide —NHCONH2NH2 groups and carbazate —OCONH2NH2 groups with preference for the former, thiosemicarbazide groups including e.g. thiosemicarbazide —NHCSNH2NH2 groups and thiocarbazate —OCSNH2NH2 groups with preference for the former, aminooxy groups e.g. —ONH2 groups (formation of hydrazone, semicarbazone, thiocarbazone, oxamine linkage structures, respectively),        b) capable of giving the linkage structure by addition to a counterpart which comprises a carbon-carbon multiple bond: thiol groups e.g. —SH (formation of thioether linkage structure),        c) capable of giving the linkage structure by addition to a counterpart which comprises a keto or aldehyde group possibly followed by elimination of H2O (formation of enamine/ketamine linkage structures) and possibly a second addition (=cyclization): β-aminothiol groups e.g. —CH(NH2)CH2SH groups (formation of thiazolidine linkage structures), and        d) miscellaneous: thiocarboxylate anionic groups e.g. —COS−.        
Groups according to (a)-(c) are preferred, in particular for in vivo formation of hydrogels since counterparts easily can be selected to give linkage structures of the appropriate stability without releasing anything or only H2O.
Preferred electrophilic groups are:                a) capable of giving the linkage structure by undergoing an addition reaction possibly followed by elimination of H2O with a counterpart which comprises an NH2— group: aldehyde and keto groups e.g. —CHO groups.        b) capable of giving the linkage structure by undergoing an addition reaction with a counterpart which comprises a thiol group: maleimide groups e.g. (CHCO)2N— groups, acrylate groups e.g. CH2═CHCOO— groups, acrylamide groups e.g. CH2═CHCONH— groups, methacrylate groups e.g. CH(CH3)═CHCOO— groups, methacrylamide groups e.g. CH(CH3)═CHCOO— groups, vinylsulphone groups e.g. CH2═CHSO2— groups,        c) capable of giving the linkage structure by ring-opening via a substitution reaction with a counterpart comprising a nucleophilic center of the kinds generally discussed above: aziridine groups, epoxide groups, reactive lactone groups and thiolactone groups (electrophilic group/center is part of a ring-structure),        d) capable of giving the linkage structure by a reaction sequence that in total is a substitution with a counterpart that comprises a nucleophilic center of the kinds generally discussed above (include formation of by-products other than H2O): α-halocarbonyl groups e.g. XCH2CONH—, XCH2COO— and XCH2CO— groups in which X is selected amongst halogens, such as Cl, Br and I), thioester groups e.g. —COSR′ groups, where R′ is lower alkyl such as C1-6 alkyl, reactive ester groups e.g. —COOR′ groups where R′ is alkoxy or aryloxy exhibiting electron-withdrawing substituents, pyridine sulphenyl groups e.g. mercaptopyridyl groups, such as (C5H4N)S— groups, β-bromoamine groups e.g. BrCH2CH(NH2)— groups.        
Groups according to (a)-(c) are preferred, in particular for in vivo formation of hydrogels. Typical linkage structures for group (a) and (b) are of the same kinds as given for nucleophilic groups (a) and (b), respectively. Typical linkage structures for group (c) are, when the nucleophilic center of the nucleophilic group exhibits an amino nitrogen or an alcoholic hydroxy oxygen, for i) aziridine groups: 1,2-diamino or 1-alkoxy-2-amino, respectively, ii) epoxide groups: 1-amino-2-hydroxy and 2-alkoxy-1-hydroxy, respectively, iii) lactone groups: hydroxy amide and hydroxy ester, respectively, and iv) thiolactone groups: mercapto amide and hydroxy amide, respectively. When thiols are reacting with aziridine groups or oxirane groups, the typical linkage structures will be 1-amino-2-alkylthiooxy or 1-hydroxy-alkylthiooxy, respectively, will be formed. For (iii) and (iv) the hydroxy and the mercapto group will be placed on the acyl moiety of the amide.
The free valence indicated in each group of the preceding paragraphs is binding to a sp-, sp2- or sp3-hybridised carbon which is part of a reactive HA- or a reactive PHP-substituent. A hydrogen H in the parent formulas of the generic groups may be replaced with a group R not negatively affecting the desired reactivity of the parent group. Thus a thiol hydrogen can not be replaced, for instance. A replacement group R is typically inert by not participating in the desired reaction of the parent group and typically is a lower alkyl possibly containing one or more structures selected amongst dialkyl ethers (—O—) or hydroxy. A lower alkyl in this context contains one, two, three, four, five up to ten sp3-hybridised carbons typically with at most one oxygen bound to one and the same carbon.
The reactive HA- and the reactive CLR-group can also be selected amongst reactive counterparts which are capable of participating in cycloaddition reactions. There are two kinds of cycloaddition reactions that are potentially of significant use in the invention                a) reactions between a diene and a dienophile as counterparts, where the diene comprises at least two conjugated multiple bonds, e.g. two carbon-carbon double bonds, and the dienophile typically exhibits a multiple bond, and        b) reactions between a multiple bond and a 1-3 dipolar group as counterparts with the basis for the dipole being a chain of three atoms comprising 1-3 heteroatoms selected amongst O, N and S and 0-2 carbons.        
Thus in variants of the invention utilizing cycloaddition reactions the pair of counterparts (i.e. the reactive HA-group and the reactive CLR-group) are selected to comprise a first counterpart that comprises a single multiple bond or at least two conjugated multiple bonds, and a second counterpart that comprises a multiple bond or an 1,3-dipolar group as defined in the preceding paragraph. Thus at least one, preferably both, of the two counterparts are selected amongst functional groups that exhibit azide structure, alkyne structure, 1,3-diene structure (e.g. 2-furan structure, 9-anthracenylmethyl structure, hexa-3,5-dien structure, cyclohexane-2,4-dien structure, cyclopentadien structure) and dienophile structure, such as benzoquinone structure.
The Hyaluronan Including its Reactive HA-Substituent
In the present invention the term “native hyaluronic acid” shall refer to an underivatized form of hyaluronic acid irrespective of being a fragment of hyaluronic acid as it exists in nature. The repetitive unit is a disaccharide consisting of D-glucuronic acid and N-acetyl D-glucosamine. Aqueous solutions of native hyaluronic acid are viscous and the viscosity increases with increasing molecular weight and concentration. In principle this also applies to the hyaluronan used in the invention which typical will restrict the use of aqueous liquid formulations containing too high concentrations of hyaluronans and hyaluronans of too high molecular weights.
The hyaluronan in the HA-formulation may be in dry form or in the form of an aqueous liquid in which the hyaluronan is dissolved. The concentration of such solutions may vary within wide ranges. More critical is the actual concentration in the aqueous liquid obtained as a consequence of the activation. Suitable concentrations in this liquid will be determined by desired rigidity, degradation rate, penetratability by a substance in the surrounding medium, ability to release an incorporated therapeutic substance, administratability (related to viscosity) etc and very much will depend on concentrations, substitution degrees, molecular weights, reactivity factors etc of both the hyaluronan and the kind of cross-linking reagent used including also mixability/administratability of the formulations (related to viscosity). Potentially useful concentrations of the hyaluronan in the liquid provided as a consequence of the activation will thus most likely be found in a wide interval, e.g. 0.1-100 mg/mL, such as 1-75 mg/mL. The preferred concentrations are found in the interval of 10-15 mg/mL, such as 12-14 mg/mL.
Suitable sizes/molecular weights of the hyaluronan of the HA-formulation will similar to the concentration depend on desired properties of the final gel which in turn in a complex way is determined by factors similar to those discussed above for the concentration. Potentially useful hyaluronans thus may be found amongst those that correspond to underivatized forms of native hyaluronic acid which have molecular weights in a wide interval, e.g. 0.2×10−2×103 kDa, such as 1×10−1×103 kDa with preference for ≧0.5×102 kDa, and/or ≧9×102 kDa, such as ≦7×102 kDa or ≦5×102 kDa where the term “correspond” means native hyaluronans which have the same number of repetitive units as the hyalurons used in the invention. The preferred range corresponds to native hyaluronic acids in the range of 0.5×102-5×102 kDa. Corresponding intervals with respect to number of repetitive units/building blocks in hyaluronans are in the interval from 5-5000 repetitive units, such as from 25-2500 repetitive units with preference for ≧125 repetitive units, and/or ≦2250 repetitive units such as ≦1750 repetitive units or ≦1250 repetitive units (based on a Mw of 400 D per disaccharide unit in Na+-form). A preferred range corresponds to 125-1250 repetitive units
A reactive HA-substituent is replacing a group which is present in native hyaluronic acid where it is directly attached to the HA-backbone (i.e. to a ring carbon). The substituent may thus be replacing the carboxy or the hydroxy group of a D-glucuronic acid moiety or the hydroxymethyl or the N-acetoamidyl group in an N-acetyl glucosamine moiety of a repetitive unit in hyaluronic acid. An alternative believed to be less preferred comprises that the reactive HA-substituent replaces an aldehyde group obtained by oxidative ring-opening of a glucuronic acid moiety of hyaluronic acid. Based on our own experience and also from what is apparent from the literature, it is believed that derivatization (=replacement) of a native carboxy group to a reactive HA-substituent will result in the most useful hyaluronans.
The hyaluronan of the HA-formulation is devoid of counterparts to the reactive HA-group, i.e. reactive groups that will cause intra-molecular cross-linking of the hyaluronan during the intended cross-linking are in essence missing in the hyaluronan used according to the invention.
A typical reactive HA-substituent provides a spacer between its reactive HA-group and the HA-backbone. The spacer typically comprises one or more bivalent groups selected amongst                a) amides for instance of carboxylic acids, such as —CXNR1—, —R1NCX—, —OCXNR1—, —R1NCXO—, —R2NCXNR1—, —CXNR1CX— where X is double-bonded oxygen, nitrogen or sulphur,        b) esters for instance of carboxylic acids —CXO—, —OCX—, —OCXO—, —R1NCO—, and —OCXNR1— where X is double-bonded oxygen, nitrogen or sulphur,        c) ethers (—X′—, where X′ is oxygen or sulphur),        d) amino (—NR1—), and        e) straight, branched or cyclic alkylene, e.g. complying with —CnH2n— or —CnH2n-2—.        
R1 and R2 in different bivalent groups of a spacer may be identical or different.
The free valence to the left in each of these bivalent groups is closer to the HA-backbone than the free valence to the right or vice versa. Each free valence is preferably binding directly to an atom carrying the free valence in i) a neighbouring bivalent group of the spacer, ii) a reactive HA-group or iii) the hyaluronan backbone. Oxygen to oxygen bonds shall be avoided. The atom in the bivalent group to which the free valence binds may thus be sp-, sp2-, or sp3-hybridised carbon, or a single bonded heteroatom (—O—, —S—, >N —) or double-bonded nitrogen.
In the alkylene chains according to (e), n is an integer within the interval of 1-10 with preference for 1-5. The alkylene chain may exhibit one or more monovalent groups directly attached to the chain and selected amongst lower alkyl, lower alkoxy, hydroxy lower alkyl, hydroxyl, amino, carboxy etc are bound. Each of these groups may thus replace a hydrogen attached to a carbon of the chain. The carbon chain of (e) may also be interrupted at one or more position by an ether oxygen or an ether sulphur. At most one heteroatom (O, N or S) is preferably bound at one and the same carbon of the bivalent alkylene of (e).
The spacer is preferably hydrophilic by having a ratio between number of heteroatoms O, N, and S and number of carbons that is ≧0.2, such as ≧0.3 or ≧0.4 (hydrophilicity ratio). The spacer is devoid of groups that will act as reactive counterparts to the reactive HA-group of the substituent. These rules for the hydrophilicity ratio are also applicable to other alkyl groups containing heteroatoms discussed in this specification.
A preferred reactive HA-substituent (and also preferred HA-spacers) is replacing a carboxy group of native hyaluronic acid and provides —CONH— directly attached to the HA-backbone via the valence to the left and to the remaining parts of the reactive HA-substituent via the other free valence.
For the same reasons as for concentrations and molecular weights/sizes, useful degrees of substitution (DS) measured (a) as number of reactive HA-substituents per repeating unit (=disaccharide unit) and/or (b) as relative amount of repeating units that carry a reactive HA-substituent may be found in a wide interval, such as ≦80%, such as ≦50% or ≦20%, with preference for ≦15% or ≦10%, with typical lower limits being 0.01% or 0.1% or 1%. A reactive HA-substituent may be dendritic, i.e. exhibiting two or more reactive HA-groups in which case the degree of substitution measured according to (a) may exceed 100%. The preferred reactive HA-substituent is non-dendritic with preferred values of DS found in the range of 1-15%, such as ≦10%.
The Cross-Linking Reagent/Polyhydroxypolymer
The cross-linking reagent in the CLR-formulation may be in dry form or in the form of an aqueous liquid in which the cross-linking reagent is dissolved. The concentration of the solutions may vary within wide ranges. More critical is the actual concentration in the aqueous liquid obtained as a consequence of the activation. Suitable concentrations in this liquid will be determined by desired rigidity, degradation rate, penetratability by a substance in the surrounding medium, ability to release an incorporated therapeutic, administratability (related to viscosity) etc, and very much will depend on concentrations, substitution degrees, molecular weights, and reactivity factors etc of both the hyaluronan and the kind of cross-linking reagent used including also mixability/administratability of the formulations (related to viscousity). Potentially useful concentrations of the cross-linking reagent in the liquid provided as a consequence of the activation will thus most likely be found in a wide interval, such as the interval of 0.1-100 mg/mL or 1-75 mg/mL. The preferred concentrations are found in the interval of 0.5-50 mg/mL, such as 1-10 mg/mL with preference for 1-3 mg/mL for polyvinyl alcohols.
As already has been discussed the cross-linking reagent is a homo-multifunctional cross-linking reagent (CLR-formulation) which                a) is lacking the backbone of the hyaluronan to be cross-linked (devoid of hyaluronan structure) and        b) provides a plurality of hydroxyl groups in the cross-linking structure introduced by the reagent.        
A preferred cross-linking reagent exhibits one, two, three, four, five or more hydroxy groups. The reagent may or may not exhibit polymeric structure.
The cross-linking reagent is devoid of counterparts to the reactive PHP-groups. In other words reactive groups that will cause intra-molecular cross-linking of the cross-linking reagent during the intended cross-linking are in essence missing in the reagent.
The cross-linking reagents believed to best are found among PHPs which exhibit a PHP-backbone with various kinds of substituents (PHP-substituents) being directly attached to the backbone. Suitable PHPs are biopolymers or are synthetic/artificial polymers.
Typical PHP-substituents are a) reactive PHP-substituents exhibiting a reactive PHP-group, b) hydroxy-containing PHP-substituents, and possibly also c) substituents neither containing a reactive PHP-group or a hydroxy group. The PHP may be a homo- or a copolymer.
Substituents according to (b) and (c) are called inert in the case they are devoid of groups that are counterparts during cross-linking conditions to the reactive PHP-groups of the cross-linking reagent because then they cannot interfere with the desired cross-linking reaction.
A PHP-backbone typically comprises a linear structure of ≧5, or more preferably ≧10 such as ≧25 monomeric units linked together one after the other. Preferably there is no branching of the polymer chain, i.e. preferred PHPs are linear and/or are devoid of cross-links. That a polymer is linear does not exclude that the polymer chain may carry projecting or pending groups of various lengths and kinds as long as such groups are not polymeric and based on the same kind of subunits as the basic chain. A PH-polymer comprises ≧5, with preference for ≧10, such as ≧25 or ≧50 hydroxyl groups and/or ≧5 monomeric subunits each of which exhibits one, two, three, four or more hydroxyl groups per unit.
A backbone of a PHP to be used in the invention thus typically comprises one or more linear structures selected amongst                a. linear polyamide backbones of identical or different monomeric subunits linked together one after the other with an amide bond (—CONR′1—,) linking two neighbouring subunits together and with corresponding monomers selected from amino carboxylic acids or combinations of a diamine with a dicarboxylic acid,        b. linear polyester backbones of identical or different monomeric subunits with an ester bond (—COO—) linking two neighbouring subunits together and with corresponding monomers selected from hydroxy carboxylic acids or combinations of a dialcohol with a dicarboxylic acid,        c. linear polyether backbones of identical or different monomeric alkylene oxide subunits with an ether bond (—O—) linking two neighbouring subunits together and a straight alkylene chain linking neighbouring ether groups together with the proviso that the chain comprises ≧2 carbons and with corresponding monomers selected from alkylene oxides.        d. linear polyvinyl backbones of identical or different monomeric C2-alkylene chain subunits —(CH2—CH2—).        e. polysaccharide backbones that are different from the hyaluronan backbone.        
Preferred backbones are believed to be found in group (d) and possibly also in group (e). Backbones of polyvinyl alcohols as used in the experimental part are considered of biggest interest. The most interesting polysaccharide is dextran.
Polymers based on backbones (a)-(c) and (e) may be called condensation polymers since H2O is formally released during the polymerisation reactions resulting in these polymers.
A group (a) and a group (b) backbone typically comprise a lower alkylene chain linking neighbouring amide groups and neighbouring ester groups, respectively, together. This chain typically is a C1-10 alkylene, and is possibly a) interrupted at one or more positions by a heteroatom-containing group, for instance ether oxygen, thioether sulphur, or amino nitrogen, and/or b) substituted with a hydroxy, hydroxy lower alkyl or lower alkyl group, such as C1-10. At most one heteroatom (O, N or S) is preferably bound to the same sp3-carbon of the alkylene chain. An important group of polyamides are those that exhibit polypeptide structure, i.e. based on hydroxy-, amino-carboxylic acids as monomers, in particular with the amino group positioned a to the carboxylic group and the hydroxy group being part of an N-hydroxyalkyl or N-hydroxyaryl group, e.g. serine, threonine, tyrosine, proline etc.
The alkylene chains in the subunits of a group (c) backbone may be identical or different between subunits, and are typically selected amongst C2-6 alkylenes, such as C2 alkylene or C3 alkylene or combinations of C2 and C3 alkylenes.
In a group (d) backbone a substituent R1 and possibly also a substituent R2 are replacing a hydrogen at the two carbons, respectively [i.e. —(CHR1—CHR2—)]. At least one of these pending substituents R1 and R2 of ≧50%, such as ≧75% or preferably ≧95%, of the subunits of the backbone exhibits a heteroatom O, N or S while the possibly remaining one of the two pending substituents may by be a lower alkyl, such as ethyl or methyl. At least one of these heteroatom-containing pending R1- and R2-substituents is in preferred variants a hydroxy group or comprises an ester oxygen, an ether oxygen, an amido nitrogen, an amino nitrogen, or a carbonyl carbon such as in keto, amido, and ester, which as indicated in preferred variants are directly attached to one of the two C atoms shown for the —(CH2—CH2—) subunit. The preferred group (d) backbone structures comprise the backbone of polyvinyl alcohol (—[CH2CH2(O—)—]n) in which the free valence of the oxygen in ≧50%, such as ≧75% or ≧85%, of the subunits binds to hydrogen or a alkyl or hydroxy alkyl containing one, two or more hydroxy groups. R1 and R2 and may differ between subunits of a polyvinyl backbone. In other polyvinyl backbone structures atoms other than oxygen may be used for linking a pending substituent R1 and R2 to the polyvinyl backbone, e.g. carbon, nitrogen and sulphur.
The reactive PHP-substituent, and, if present, inert hydroxyl-containing PHP-substituents and other PHP-substituents are attached directly to atoms of a selected backbone structure.
Suitable polyhydroxypolymers comprising a polyvinyl backbone (d above) are typically found amongst polymers containing one, two or more different monomeric units deriving from hydroxyalkyl acrylates and methacrylates, N-hydroxyalkyl acryl- and n-hydroxyalkyl methacrylamides, hydroxyalkyl vinyl ethers, vinyl esters etc. Polyvinyl alcohols are typically obtained by partial hydrolysis of polyvinyl esters meaning that polyvinyl alcohols that are preferred in the invention typically exhibit residual amounts of ester groups (≦10% or ≦5%). Hydroxyalkyl comprises alkyl groups in which there are one or more hydroxy groups possibly in combination with one or more ether groups with the proviso that there preferably is at most one oxygen bound to one and the same carbon.
A typical reactive PHP-substituent provides a spacer between its reactive PHP-group and the PHP-backbone. The spacer typically comprises one or more bivalent groups selected amongst the same bivalent groups as those given for a spacer of a reactive HA-substituent.
A preferred reactive PHP-substituent comprises an ester group between the PHP-backbone and the reactive PHP-group. Typical ester functions are selected amongst —OCO—, —OCOO— and —OCONR1— where the left free valence preferably is closer to the PHP-backbone than the right free valence. In preferred hydroxypolymers the left free valence is attached directly to a carbon of the PHP-backbone.
Suitable sizes/molecular weights of the polyhydroxypolymer will similar to the concentration depend on desired properties of the final gel which in turn in a complex way is determined by factors similar to those discussed above for the concentration of the cross-linking reagent Potentially useful polyhydroxypolymers for use in a PHP-formulation may thus be found amongst those in which the number (mean values) of monomeric units are at least 20, 100, 200, 300, 500, 1000, or 2000, and at most 100, 200, 300, 500, 1000, 2000 or 20000 (with a lower limit of an interval always being smaller than the upper limit). Preferred numbers of monomeric units are found in the interval of 200-600 which in particular applies to the polyvinylalcohol used in the experimental part.
For the same reasons as for concentrations and molecular weights, useful degrees of substitution (DS) measured (a) as number of reactive PHP-substituents per repeating unit (=disaccharide unit) and/or (b) as relative amount of repeating units that carry a reactive PHP-substituent may be found in a wide interval, such as ≦80%, such as ≦50% or ≦20%, with preference for ≦15% or ≦10%, with typical lower limits being 0.01% or 0.1% or 1%. A reactive PHP-substituent may be dendritic, i.e. exhibiting two or more reactive PHP-groups in which case the degree of substitution measured according to (a) may exceed 100%. The preferred reactive PHP-substituent is non-dendritic with preferred values of DS found in the range of 1-15%, such as ≦10%.
In the preferred kinds of polyvinyalcohols a repetitive unit can carry at most one reactive PHP-substituent that may carry one or more reactive PHP-group. Examples of potentially suitable PHPs that may carry more than one reactive PHP-substituent per repetitive unit are found amongst polysaccharides, e.g. dextrans and polyvinylalcohols derivatized to exhibit hydroxyalkylether groups in which the hydroxyalkyl groups carries a plurality of hydroxy groups. Other potentially interesting examples are poly(alkyl vinyl ether)polymers in which a plurality of the alkyl groups carries a plurality of hydroxy groups and possibly with the carbon chain of the alkyl group being interrupted at one or more positions by an ether bond with the proviso that preferably at most one oxygen is bound at one and the same sp3-hybridised carbon.
Other Features of the Formulations of the Compositions and Vehicles for the Composition.
The composition of the invention typically comprises a buffer system that shall provide a physiologically acceptable pH in the aqueous liquid in which the cross-linking reaction and hydrogel formation are to take place. Physiological acceptable in this context means acceptable for the individual to whom the composition is to be administered and includes pH values that are outside what is normally contemplated for healthy individuals. Thus suitable pH-values for the invention are found within the pH-range of 4.5-9, with preference for 5.5-8.5. These ranges typically apply for the above-mentioned aqueous liquid and possibly also for one or more of the formulations given above, e.g. the HA- and/or the CLR-formulation. Buffering components of the buffer system selected may be present either in one or both of the HA- and CLR-formulations and/or in a separate buffer formulation. The buffering components may differ between the formulations of a composition both with respect to kinds and relative amounts. It follows that the pH-value provided by buffering components in one of the formulations may be different from the pH-value provided by the buffering components in the other formulation. The pH-value may be physiologically unacceptable in one or both of the HA- and the CLR-formulation as long as mixing of the two formulations with each other possibly supplemented with buffer components from a separate buffer formulation will result in physiologically acceptable pH conditions in the final aqueous solution after activation is completed. Physiologically unacceptable pH-conditions may be appropriate and even advantageous in a single formulation, among others for stability reasons of particular variants and combinations of reactive counterparts, for instance.
The composition may also contain various kinds of salts to properly provide acceptable ionic strengths and the like in the aqueous solution in which cross-linking and hydrogel formation are to take place. Such salts may be present in either one or both of the HA- and CLR-formulations and/or in a separate formulation, for instance.
One, two or more of the different formulations of the composition may be in aqueous liquid form or in solid form, e.g. a solid form which is possible to reconstitute to aqueous liquid form. Solid forms comprise powder forms, dehydrated forms such as lyophilized forms, spray-dried forms, air dried forms, etc with reconstitution to aqueous liquid form (solution) typically taking place during the activation of the composition.
The composition may also comprise a compartmentalized vehicle in which there for each formulation of the composition typically is a separate compartment with an outlet conduit that ends in an outlet of the vehicle. The outlet of the vehicle may be common or separate for two or more compartments which in particular applies to the compartments for the HA- and the CLR-formulations. The outlet of these preferred vehicles shall thus be                a) common for the formulations, in particular the HA- and the CLR-formulations thereby allowing administration of a mixture of the formulations, i.e. an activated form of the composition (in aqueous liquid form) as required by the intended use and discussed elsewhere in this specification, or        b) separate for the formulations permitting parallel administration of the formulations with the final step in the activation (mixing) taking place at the location to which they have been administered.        
These kinds of vehicles are preferably in the form of a compartmentalized syringe of the kind well-known in the field and used in the experimental part. The compartments of the vehicle may be replaceable permitting compartments of different volumes facilitating the use different relative amounts of the formulations of the composition, for instance the HA- and CLR-formulations.
Other vehicles are also possible, for instance separate vessels (ampoules, bottles etc) for the different formulations. These vessels may be storage vessels from which aliquots are transferred to the corresponding compartments of a compartmentalized vehicle.
The HA- and the CLR-formulation of the inventive composition are typically delivered to the customer or end user either separately or together. There are typically attached a package insert and/or a manual of use referring to the HA- and the CLR-formulation and/or a use as discussed elsewhere in this specification.
The composition of the invention may also contain one or more formulations containing a population of particles (particle formulations). The particles may or may not be bioactive. A particle formulation may coincide with one or more of the formulations discussed above or is a separate formulation, with preference for either one or both of the HA- and CLR-formulations also being a particle formulation. The particles should at least be dispersible in the aqueous liquid which contains both the hyaluronan and the cross-linking reagent and is obtained as a consequence of activation of the composition.
A particle formulation may be devoid of aqueous liquid with the particles being present as a dispersible powder or a dehydrated form, e.g. of the kinds discussed above for other formulations. The preferred particle formulations are similar to the preferred HA- and CLR-formulations in the sense that both of them are based on the presence of an aqueous liquid. The particles are during storage of the formulation maintained in dispersed form or alternatively settled but dispersible.
Dispersible particles populations are typically nanosized (nanoparticulate), i.e. have a mean size in the nm-range, i.e. ≦5000 nm, such as ≦1000 nm with typical lower limits being 0.1 or 0.5 nm.
Particles of the composition may act as a filler agent for the hydrogel to be formed and will then typically improve the mechanical strength of the gel. Filler particles may be based on organic material and/or inorganic material. Suitable inorganic material is typically biocompatible, such as apatite, titanium etc.
The particles may be bioactive, e.g. be a therapeutically active. In most instances bioactive particles may also act as filler agents.
Typical useful bioactive particles with characteristics as outlined above may contain                a) bioactive inorganic material, e.g. Ca2+ in salt form with phosphate as counter-ion for instance, such as dispersible nanoparticulate forms of apatite and other variants of calcium phosphate, and/or        ii) bioactive organic material, e.g. of bioorganic origin such as immunogenic bio-organic material deriving from unicellular organisms, such as bacteria, fungi etc, viruses, plants and animals        iii) organic and/or inorganic material to which a bioactive substance of the kinds discussed below is immobilized.        
The composition of the invention may also contain a bioactive substance that is not in particle form but in soluble/dissolved form. The most important such substances are therapeutically active compounds such as drugs. They typically exhibit at least one structure selected amongst peptide structures (including polypeptide structures (such as oligopeptide structures) and protein structures), nucleotide structures (including i.a. polynucleotide structures), carbohydrate structures, steroid structures, lipid structures, antioxidant structures, vitamin structures etc and structures of drugs in the form of low molecular weight organic compounds of molecular weights<5 kDa, such as <2 kDa or <1 kDa with a typical lower limit of 0.1 kDa.
The bioactive substance may be free to dissociate from the hydrogel without need for breaking a covalent bond between the substance and the cross-linked network of the hydrogel. This does not exclude that there may be variants in which the bioactive substance is covalently attached to the hydrogel and not released until a covalent bond immobilizing the substance to the hydrogel is broken.
In certain other variants the bioactive substance may be present in the HA-formulation or in the PHP-formulation covalently immobilized or covalently immobilizable to the hyaluronan or the PHP. If the bioactive substance is immobilizable, immobilization can take place prior to, during and/or subsequent to hydrogel formation, e.g. by reacting with excessive amounts of a counterpart group. In most of these variants the major route for release of the substance is by degradation in vivo of the hydrogel.
Growth factors are typical bioactive substances which exhibit peptide structure. A growth factor typically has a relatively high molecular weight, such as ≧2 kDa, such as ≧5 kDa or ≧10 kDa, induces proliferation and/or differentiation of cells, and/or preferably is active locally when present in vivo. Typical examples are growth factors of the TGF-family including osteoinductive growth factors such as various bone morphogenetic proteins, for instance BMP-2, -4, -6, -7, -12, and other growth factors such as vascular endothelium growth factors (VEGF), epidermal growth factors (EGF), fibroblast growth factors (FGF), nerve growth factors (NGF), platelet derived growth factor (PDGF), insulin-like growth factors, such as IGF-1.
Important tissue generation applications of hydrogels prepared from the compositions of the invention are:                i) Hydrogels containing osteoinductive growth factors may be used to improve fracture healing or heal bone defects including spinal fusion procedures. The use for enhanced osseointegration of implants, such as dental and orthopaedic implants, is also included        ii) Hydrogels with a particle additive, such as hydroxyapatite and/or tricalcium phosphate, may enhance the amount of bone formation. The hydrogel with particle additives may function without osteoinductive BMPs as bone conductive agent for the healing of bone fractures, bone defects, and improved osseointegration of bone implants. Hydrogels containing a particle additive, and osteoinductive BMPs are likely to allow osteoconduction and/or osteoinduction.        iii) Hydrogels containing particle additives and with or without osteoinductive growth factors, may not only be beneficial in the treatment of bone defects but also in generalized bone disease including osteoporosis. This kind of composition may be injected into bones.        iv) Hydrogels with or without hydroxyapatite particles, containing members in the BMP-family of growth factors, including BMP-2, -4, -6, -7, -12, -13, and growth and differentiation factor-5 (GDF-5) may be therapeutically beneficial for healing of localized or generalized cartilage defects and tendon repair.        v) Hydrogels containing purified or recombinant growth factors including VEGF, EGF, FGF, NGF, PDGF, IGF-1 etc may be used for local administration for obtaining a local cellular response and tissue regeneration including vascular formation and dermal/epidermal healing.        
Chemotherapeutics drugs (bioactive substances) of the type discussed above may be incorporated in the composition and be beneficial in cancer treatment by local and/or systemic sustained release of the drug.
A bioactive substance of the type discussed above, e.g. a bioactive peptide (typically of oligopeptide structure), may be incorporated in an inventive composition intended for local or systemic in vivo administration and response.
A bioactive substance/drug of the type discussed above, for instance an antagonist such as a TNF-alpha antagonist, may be incorporated in an inventive composition intended for sustained release and therapeutic effects of the bioactive substance in vivo.
A bioactive substance of the type discussed above, such as a drug which may be a hormone or a steroid, e.g. a growth hormone, insulin, corticosteroid etc, may be incorporated in an inventive composition intended for delivery and slow release in vivo for achieving a therapeutic effect.
An immunogen may be incorporated in an inventive composition intended to be used as a vaccine adjuvant for immunization in order to accomplish antibody/humoral and cell-mediated immune responses.
Nucleic acids such as cDNA or siRNA may be incorporated in an inventive composition intended for research and/or therapeutic purposes.
A vitamin may be incorporated in an inventive composition intended to be used for administration of vitamins by injection. The vitamin-depot may allow systemic release of antioxidants (vitamin C and E) and/or specifically target immune cells for instance lymphocytes leading to improved anti-viral, anti-bacterial, and/or anti-cancer effects mediated by the immune system.
A second main aspect of the invention is the composition of the first aspect for use in vivo or ex vivo as a support matrix. This kind of uses encompasses applications in vivo using the support matrix (=the hydrogel formed) in tissue generation including both tissue generation and tissue engineering, as a sustain release storage for a bioactive substance of the kinds discussed above (e.g. a therapeutics, an immunogen etc), in cosmetic surgery (breast implants, tissue augmentation/anti-wrinkling etc), in keeping tissue together (tissue gluing), in keeping tissue apart (tissue separator), for promoting the presentation of a bioactive substance to receptors in vivo (e.g. as an adjuvant as discussed above) etc.
The use aspect also comprises the use of a HA-formulation and a CLR-formulation of the kinds discussed above for the manufacture of a composition of the first aspect to be used for the treatment of an individual considered to be in need for the support matrix obtained by activation of the composition and formation of the corresponding hydrogel in vivo at the desired location within the individual.
A third main aspect of the invention is a method for providing in vivo a support matrix for one or more of the purposes discussed above to an individual or an organ deemed to be in need for the support matrix for these purposes. The method comprises the steps of:                i) providing a composition as discussed for the first aspect, preferably with said formulations being in aqueous liquid form,        ii) activating said composition to give a common aqueous liquid containing said hyaluronan and said cross-linking reagent, and, if present in the composition provided in step (i), said buffer, said particles and/or said bioactive substance,        iii) administering the liquid obtained in step (ii) to the location within said individual or said organ so that a hydrogel will be formed at said location containing, if present in the composition provided in step (i), said particles and/or said bioactive substance.        
The method may comprise optional steps. One such step may be a fourth step comprising administering a bioactive substance to the gel formed in vivo during step (iii) if said individual or said organ is deemed in need for this substance. This step in particular applies if a desired bioactive substance was not including in the composition provided in step (i) or if an additional dose is needed of a substance included from the beginning.